Prodrugs of excitatory amino acids

ABSTRACT

This invention relates to synthetic excitatory amino acid prodrugs according to formula (I) and processes for their preparation. The invention further relates to methods of using, and pharmaceutical compositions comprising, the compounds for the treatment of neurological disorders and psychiatric disorders.

[0001] This invention relates to synthetic excitatory amino acidprodrugs (and their pharmaceutically acceptable salts) and processes fortheir preparation. The invention further relates to methods of using,and pharmaceutical compositions comprising, the compounds for thetreatment of neurological disorders and psychiatric disorders.

[0002] Treatment of neurological or psychiatric disorders, such asanxiety disorder, have been linked to selective activation ofmetabotropic excitatory amino acid receptors such as(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, also known asLY354740, which is disclosed in U.S. Pat. No. 5,750,566 (the '566patent) issued May 12, 1998 is an active MGLUR2 receptor agonist, CNSDrug Reviews, 5, pgs. 1-12 (1999).

[0003] The present invention provides for a prodrug form of LY354740,which enhances the in vivo potency of LY354740, producing higher oralexposure of the parent compound. In addition, when compounds of thepresent invention are administered, no circulating level of prodrug wasdetected with high in vitro bioconversion to the parent molecule.Further, the peptide prodrugs are stable under all ranges of pH and arenontoxic. Compounds of the present invention represent the best approachfor maintaining LY354740-like safety and efficacy in humans withincreased oral bioavailability. Preclinical studies with,(1S,2S,5R,65)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride, the compound of the present invention, has showngreatly enhanced oral potency in the treatment of anxiety without theattendant problems of toxicity, instability at desired pH ranges and lowin vivo conversion.

[0004] Accordingly, the present invention provides a compound of formulaI

[0005] wherein

[0006] R¹³, R¹⁴ and R¹⁷ are hydrogen;

[0007] or a pharmaceutically acceptable salt thereof.

[0008] Compounds of the invention have been found to be useful prodrugsfor LY354740 a selective agonist of metabotropic glutamate receptors andare therefore useful in the pharmaceutical treatment of diseases of thecentral nervous system such as neurological diseases, for exampleneurodegenerative diseases, and as antipsychotic, anxiolytic,drug-withdrawal, antidepressant, anticonvulsant, analgesic andanti-emetic agents.

[0009] It will be appreciated that the compounds of formula (I) containat least four asymmetric carbon atoms, three being in the cyclopropanering and one being at the α-carbon of the amino acid group. Accordingly,the compounds of the invention may exist in and be isolated inenantiomerically pure form, in racemic form, or in a diastereoisomericmixture.

[0010] The amino acid moiety preferably has the natural amino acidconfiguration, i.e. the L-configuration relative to D-glycerol aldehyde.

[0011] The present invention includes pharmaceutically acceptable saltsof the compound of formula I. These salts can exist in conjunction withthe acidic or basic portion of the molecule and can exist as acidaddition, primary, secondary, tertiary, or quaternary ammonium, alkalimetal, or alkaline earth metal salts. Generally, the acid addition saltsare prepared by the reaction of an acid with a compound of formula I.The alkali metal and alkaline earth metal salts are generally preparedby the reaction of the hydroxide form of the desired metal salt with acompound of formula I.

[0012] Some particular salts provide certain formulation advantages dueto their crystalline form. Non-crystalline forms of compounds may beamorphous and hygroscopic. Crystalline forms of pharmaceutical compoundsare sometimes more desirable because they are not amorphous.

[0013] A particular pharmaceutically acceptable salt of the peptide offormula I is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride salt.

[0014] Another particular pharmaceutically acceptable salt of thepeptide of formula I is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methane sulfonate salt.

[0015] Acids commonly employed to form such salts include inorganicacids, for example hydrochloric, hydrobromic, nitric, sulphuric orphosphoric acids, or with organic acids, such as organic carboxylicacids, for example, glycollic, maleic, hydroxymaleic, fumaric, malic,tartaric, citric, salicyclic, o-acetoxybenzoic, or organic sulphonic,2-hydroxyethane sulphonic, toluene-p-sulphonic, methane-sulfonic ornaphthalene-2-sulphonic acid.

[0016] In addition to pharmaceutically-acceptable salts, other salts areincluded in the invention. They may serve as intermediates in thepurification of compounds or in the preparation of other, for examplepharmaceutically-acceptable, acid addition salts, or are useful foridentification, characterization or purification.

[0017] A variety of physiological functions have been shown to besubject to influence by excessive or inappropriate stimulation ofexcitatory amino acid transmission. The formula I compounds of thepresent invention are believed to have the ability to treat a variety ofneurological disorders in mammals associated with this condition,including acute neurological disorder such as cerebral deficitssubsequent to cardiac bypass surgery and grafting, stroke, cerebralischemia, spinal cord trauma, head trauma, perinatal hypoxia, cardiacarrest, and hypoglycemic neuronal damage. The formula I compounds arebelieved to have the ability to treat a variety of chronic neurologicaldisorders, such as Alzheimer's disease, Huntington's Chorea, amyotrophiclateral sclerosis, AIDS-induced dementia, ocular damage and retinopathy,cognitive disorders, and idiopathic and drug-induced Parkinson's. Thepresent invention also provides methods for treating these disorderswhich comprises administering to a patient in need thereof an effectiveamount of a compound of formula I or a pharmaceutically acceptable saltthereof.

[0018] The formula I compounds of the present invention treat a varietyof other neurological disorders in patients that are associated withglutamate dysfunction, including muscular spasms, convulsions, migraineheadaches, urinary incontinence, pain, premenstrual dysphoric disorder(PDD), psychosis, (such as schizophrenia), drug tolerance and withdrawal(such as nicotine, opiates and benzodiazepines), anxiety and relateddisorders, emesis, brain edema, chronic pain, and tardive dyskinesia.The formula I compounds are also useful as antidepressant and analgesicagents. Therefore, the present invention also provides methods fortreating these disorders which comprise administering to a patient inneed thereof an effective amount of the compound of formula I, or apharmaceutically acceptable salt thereof.

[0019] A compound of Formula I may be made by a process which isanalogous to one known in the chemical art for the production ofstructurally analogous heterocyclic compounds or by a novel processdescribed herein. Such processes and intermediates useful for themanufacture of a compound of Formula I as defined above are provided asfurther features of the invention and are illustrated by the followingprocedures in which, unless otherwise specified, the meanings of thegeneric radicals are as defined above.

[0020] (A) For a compound of formula I in which R¹³, R¹⁴, and R¹⁷ arehydrogen (a di-acid), deprotecting the amine group of a compound offormula I

[0021] where R¹⁷ is tert-butoxy carbonyl or a nitrogen protecting group,with an acid as described in the General Procedures for Examples 3 and4.

[0022] (B) For a compound of formula I in which R¹³ and R¹⁴ are bothhydrogen (a di-acid), deprotecting a compound of formula I where R¹³ andR¹⁴ are not both hydrogen as described in Scheme 2.

[0023] (C) For a compound of formula I in which R¹³ and R¹⁴ are not bothhydrogen, amidating a compound of formula II

[0024] with a corresponding amino acid of formula III.

HOOCCHCH₃NHR¹⁷   III

[0025] in which p is O or any integer from 1-10 and R¹⁷ is tert-butoxycarbonyl or a nitrogen-protecting group as described in the GeneralProcedure for Example 1.

[0026] (D) For a compound of formula II where R¹³ and R¹⁴ are nothydrogen, where R¹³ and R¹⁴ may be a carboxy-protecting ester group (adi-ester), esterifying a compound of formula II where R¹³ and R¹⁴ areboth hydrogen (a di-acid).

[0027] (E) For a compound of formula II in which R¹³ and R¹⁴ are nothydrogen (a di-ester), deprotecting a compound of formula IV

[0028] where R^(m) is a nitrogen protecting group, as described inPreparation 2.

[0029] (F) For a compound of formula II where R¹³ and R¹⁴ are not bothhydrogen (a di-ester), esterifying a compound of formula IV, asdescribed in Preparation 2.

[0030] (G) For a compound of formula IV where R¹³ and R¹⁴ are bothhydrogen (a di-acid), protecting the amine group of a compound offormula II as described in Preparation 1.

[0031] The term “nitrogen protecting group,” as used herein, refers tothose groups intended to protect or block the nitrogen group againstundesirable reactions during synthetic procedures. Choice of thesuitable nitrogen protecting group used will depend upon the conditionsthat will be employed in subsequent reaction steps wherein protection isrequired, as is well within the knowledge of one of ordinary skill inthe art. Commonly used nitrogen protecting groups are disclosed in T. W.Greene and P. G. M. Wuts, Protective Groups In Organic Synthesis, 2^(nd)Ed. (John Wiley & Sons, New York (1991)).

[0032] The term “carboxy-protecting group” as used herein refers to oneof the ester derivatives of the carboxylic acid group commonly employedto block or protect the carboxylic acid group while reactions arecarried out on other functional groups of the compound. Particularvalues include, for example, methyl, ethyl, tert-butyl, benzyl,methoxymethyl, trimethylsilyl, and the like. Further examples of suchgroups may be found in T. W. Greene and P. G. M. Wuts, Protecting Groupsin Organic Synthesis, 3 rd. Ed. (John Wiley & Sons, N.Y. (1999)). Theester is decomposed by using a conventional procedure which does notaffect another portion of the molecule.

[0033] Whereafter, for any of the above procedures, when apharmaceutically acceptable salt of a compound of Formula I is required,it is obtained by reacting the acid of Formula I with a physiologicallyacceptable base or by reacting a basic compound of Formula I with aphysiologically acceptable acid or by any other conventional procedure.

[0034] The term “C₁-C₁₀ alkyl” represents a straight, branched, orcyclic alkyl chain having from one to ten carbon atoms.

[0035] The term “C₂-C₁₀ alkenyl” represents straight or branchedunsaturated alkyl chains having from two to ten carbon atoms, and havingone or more carbon-carbon double bond, such as, dienes and trienes. Thisgroup also includes both E and Z isomers.

[0036] The term “aryl” represents groups such as phenyl, substitutedphenyl, and naphthyl. The term “arylalkyl” represents a C₁-C₄ alkylgroup bearing one or more aryl groups.

[0037] The term “affecting” refers to a formula I compound acting as anagonist at an excitatory amino acid receptor. The term “excitatory aminoacid receptor” refers to a metabotropic glutamate receptor, a receptorthat is coupled to cellular effectors via GTP-binding proteins. The term“cAMP-linked metabotropic glutamate receptor” refers to a metabotropicreceptor that is coupled to inhibition of adenylate cyclase activity.

[0038] The term “neurological disorder” refers to both acute and chronicneurodegenerative conditions, including cerebral deficits subsequent tocardiac bypass surgery and grafting, cerebral ischemia (for examplestroke resulting from cardiac arrest), spinal cord trauma, head trauma,Alzheimer's Disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, perinatal hypoxia, hypoglycemic neuronal damage,ocular damage and retinopathy, cognitive disorders, idiopathic anddrug-induced Parkinson's Disease. This term also includes otherneurological conditions that are caused by glutamate dysfunction,including muscular spasms, migraine headaches, urinary incontinence,drug tolerance, withdrawal, and cessation (i.e. opiates,benzodiazepines, nicotine, cocaine, or ethanol), smoking cessation,emesis, brain edema, chronic pain, sleep disorders, convulsions,Tourette's syndrome, attention deficit disorder, and tardive dyskinesia.

[0039] The term “psychiatric disorder” refers to both acute and chronicpsychiatric conditions, including schizophrenia, anxiety and relateddisorders (e.g. panic attack and stress-related cardiovasculardisorders), depression, bipolar disorders, psychosis, and obsessivecompulsive disorders.

[0040] A particular aspect of the present invention includes a methodfor affecting the cAMP-linked metabotropic glutamate receptors in apatient, which comprises administering to a patient requiring modulatedexcitatory amino acid neurotransmission a pharmaceutically-effectiveamount of a compound of formula I.

[0041] Another particular aspect of the present invention includes amethod of administering an effective amount of a compound of formula II,where R¹³ and R¹⁴ are both hydrogen (a di-acid), which comprisesadministering to a patient requiring modulated excitatory amino acidneurotransmission a pharmaceutically effective amount of a compound offormula I.

[0042] Another particular aspect of the present invention includes amethod for treating a psychiatric disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of formula I.

[0043] Another particular aspect of the present invention includes amethod for treating a neurological disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of formula I.

[0044] A preferred method for treating a psychiatric disorder in apatient comprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid psychiatric disorder is schizophrenia, anxiety and relateddisorders, depression, dipolar disorders, psychosis, and obsessivecompulsive disorders.

[0045] A preferred method for treating a neurological disorder in apatient comprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid neurological disorder is cerebral deficits subsequent to cardiacbypass and grafting; cerebral ischemia; spinal cord trauma; head trauma;Alzheimer's Disease; Huntington's Chorea; amyotrophic lateral sclerosis;AIDS-induced dementia; perinatal hypoxia; hypoglycemic neuronal damage;ocular damage and retinopathy; cognitive disorders; idiopathic anddrug-induced Parkinsons' Disease; muscular spasms; migraine headaches;urinary incontinence; drug tolerance, withdrawal, and cessation; smokingcessation; emesis; brain edema; chronic pain; sleep disorders;convulsions; Tourette's syndrome; attention deficit disorder; andtardive dyskinesia.

[0046] A more preferred method for treating a psychiatric disorder in apatient comprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid psychiatric disorder is anxiety and related disorders.

[0047] A more preferred method for treating a neurological disorder in apatient comprises administering to the patient in need thereof apharmaceutically-effective amount of a compound of formula I whereinsaid neurological disorder is drug tolerance, withdrawal, and cessation;or smoking cessation.

[0048] An additional aspect of the present invention is a compound offormula I, or a pharmaceutically acceptable salt thereof, for use as apharmaceutical.

[0049] Another aspect of the present invention includes the use of acompound of formula I, or a pharmaceutically acceptable salt thereof,for the manufacture of a medicament for treating neurological orpsychiatric disorders.

[0050] As used herein the term “effective amount” refers to the amountor dose of the compound, upon single or multiple dose administration tothe patient, which provides the desired effect in the patient underdiagnosis or treatment.

[0051] An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances. For example, a typicaldaily dose may contain from about 25 mg to about 300 mg of the activeingredient. The compounds can be administered by a variety of routesincluding oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, bucal or intranasal routes. Alternatively, the compoundmay be administered by continuous infusion.

[0052] As used herein the term “patient” refers to a mammal, such as amouse, guinea pig, rat, dog or human. It is understood that thepreferred patient is a human.

[0053] The term “treating” (or “treat”) as used herein includes itsgenerally accepted meaning which encompasses prohibiting, preventing,restraining, and slowing, stopping, or reversing progression of aresultant symptom. As such, the methods of this invention encompass boththerapeutic and prophylactic administration.

[0054] If not commercially available, the necessary starting materialsfor the above procedures may be made by procedures which are selectedfrom standard techniques of organic and heterocyclic chemistry,techniques which analogous to the syntheses of known, structurallysimilar compounds, and the procedures described in the Examples,including novel procedures.

[0055] A further aspect of the present invention provides for a methodof administering an effective amount of a compound of formula II, whereR¹³ and R¹⁴ are both hydrogen (a di-acid), which comprises administeringto a patient requiring modulated excitatory amino acid neurotransmissiona pharmaceutically-effective amount of a compound of formula I.

[0056] Compounds of formula I are converted via enzymatic or hydrolyticprocess in vivo, to form compounds of formula II, where R¹³ and R¹⁴ areboth hydrogen (a di-acid), as shown in Scheme 1 below.

[0057] In particular, a crystalline form of a compound of formula I maybe prepared according to the route outlined in Scheme 2 below in whicheach of R¹³ and R¹⁴, respectively, represents a value defined for thegroups R¹³ and R¹⁴. The process described in Scheme 2 is a synthesismethod for the preparation of a crystalline hydrochloride salt form of acompound of formula I and a methanesulfonate salt form of a compound offormula I.

[0058] In scheme 2 above, the monohydrate of II, where R¹³ and R¹⁴ areboth hydrogen (a di-acid), is treated with thionyl chloride and methanolaffording the corresponding di-ester of II. Alternatively, catalytichydrochloric acid may be used in place of thionylchloride. The di-ester,formula II, is amidated with a compound of formula III usingdicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) or isobutylchloroformate as a coupling agent to afford a di-ester protectedpeptidyl compound of formula I. This transformation could also beachieved using the acid chloride or by using a variety of other peptidecoupling reagents, for example, diphenyl chlorophosphate and2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),bis(2-oxo-3-oxazolidinyl)phosphinic chloride andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.

[0059] The hydrolysis of the di-ester protected peptidyl compound offormula I with a suitable base such as lithium hydroxide or sodiumhydroxide in THF affords the di-acid protected peptidyl compound offormula I, where R13 and R14 are both hydrogen (a di-acid). The di-acidprotected peptidyl compound of formula I may be deprotected with amineral or organic acid in a suitable solvent. Such conditions mayproduce the corresponding acid salt of the di-acid peptidyl compound offormula I as an amorphous solid or, directly, a crystalline solid. Inthe case of an amorphous solid, subsequent crystallization may occurfrom suitable solvents. For example, a di-acid protected peptidylcompound of formula I when treated hydrogen chloride gas in ethylacetate provides the deprotected hydrochloride salt as an amorphoussolid. The amorphous hydrochloride compound may then be crystallizedfrom acetone and water to afford the crystalline hydrochloride saltcompound of formula I. In the case of a crystalline solid which isformed directly, filtration of the reaction mixture may afford thecrystalline salt. The zwitterionic compound of formula I is afforded bytreatment of the crystalline hydrochloride salt of formula I with sodiumhydroxide. It will be appreciated by one of ordinary skill in the artthat a compound of formula I may be prepared in one procedure where theindicated intermediates are not isolated.

[0060] The ability of compounds to modulate metabotropic glutamatereceptor function may be demonstrated by examining their ability toinfluence either cAMP production (mGluR 2, 3, 4, 6, 7 or 8) orphosphoinositide hydrolysis (mGluR 1 or 5) in cells expressing theseindividual human metabotropic glutamate receptor (mGluR) subtypes. (D.D. Schoepp, et al., Neuropharmacol., 1996, 35, 1661-1672 and 1997, 36,1-11) .

[0061] The ability of formula I compounds to treat anxiety or a relateddisorder may be demonstrated using the well known fear potentiatedstartle and elevated plus maze models of anxiety described respectivelyin Davis, Psychopharmacology, 62:1;1979 and Lister, Psychopharmacol,92:180-185; 1987

In Vitro Receptor Binding

[0062] To study the ability to affect receptor binding of compounds ofthe present invention in comparison to LY354740, displacement of a highaffinity mGluR2 antagonist radioligand [³H]LY341495 to cell membranesfrom human mGluR2, human mGluR3, and native rat brain tissues wasdetermined. (See, Ornstein P. L., Arnold M. B., Bleisch T. J., Wright R.A., Wheeler W. J., and Schoepp D. D., [³H]LY341495, a highly potent,selective and novel radioligand for labeling group II metabotropicreceptors. Bioorg. Med. Chem. Lett. 8: 1919-1922 (1998); and Johnson B.G., Wright R. A., Arnold M. B., Wheeler W. J., Ornstein P. L., andSchoepp D. D., [³H]LY341495 as a novel rapid filtration antagonistradioligand for group II metabotropic receptors: Characterization ofbinding to membranes of mGlu receptor subtype expressing cells.Neuropharmacology 38: 1519-1529 (1999))

[0063] As shown in Table 1 below, LY354740 displaced [³H]LY341495binding to rat forebrain membranes with a potency similar to thatobserved in human recombinant receptors. In contrast, the compound offormula I did not appreciably displace [³H]LY341495 binding to ratforebrain membranes at up to 10,000 nM. TABLE 1 Comparison of receptorbinding of compounds of the present invention with LY354740 Displacementof ³H-LY341495 binding Receptor (Ki, nM) (mean ± S.E., N = 3)Preparation LY354740 Formula I Human mGluR2  84.7 ± 11.1 >10,000 HumanmGluR3 125.6 ± 4.8 >10,000 Rat Forebrain  80.0 ± 7.3 >10,000

In Vivo Actions in Rat Fear Potentiated Startle Anxiety Model

[0064] To study the oral potencies of compounds of the present inventionin comparison to LY354740 in an mGlu2/3 receptor linked therapeuticanimal model, studies in the rat, fear-potentiated startle assay wereperformed. This model was specifically chosen, as it is highly sensitiveto mGlu2/3 agonists such as LY354740 and compounds of the presentinvention. (See, Helton D. R., Tizzano J. P., Monn J. A., Schoepp D. D.,and Kallman M. J., Anxiolytic and side-effect profile of LY354740: Apotent, high selective, orally active agonist for group II metabotropicglutamate receptors, J. Pharmacol. Exp. Ther. 284: 651-660 (1998)). Toverify that the actions of a compound of formula I in this model weremGlu2/3 receptor mediated, as has been shown previously for LY354740(See, Tizzano, J. P., Griffey K. I., Ornstein P. L., Monn J. A., andSchoepp D. D., Actions of mGlu receptor agonists on fear-conditioningversus fear-expression in rats, Neuropharmacology, 38:A45 (#144)(1999)), the ability of LY341495 (an mGlu2/3 receptor antagonist) (See,Kingston A. E., Ornstein P. L., Wright R. A., Johnson B. G., Mayne N.G., Burnett J. P., Belagaje R., Wu S., and Schoepp D. D., LY341495 is ananomolar potent and selective antagonist for group II metabotropicglutamate receptors, Neuropharmacology, 37: 1-12 (1998)) to blockcompound-mediated suppression of fear-potentiated startle was alsodetermined. As a positive control in each experiment, diazepam (0.6mg/kg i.p.) was used. All experiments were performed in fed rats.

[0065] In the fear potentiated startle model, animals are exposed to aneutral stimulus such as light (conditioned stimulus) with an aversivestimulus such as a shock (unconditioned stimulus). Followingconditioning, when the animals are presented with a loud acousticstimulus, larger startle responses are elicited when the startlestimulus is preceded by light.

[0066] Diazepam and buspirone hydrochloride, which are clinically provenanxiolytics, are effective at reducing the fear (increased startleresponse) associated with the presentation of light in the fearpotentiated startle model, and in reducing the fear of open spaces inthe elevated plus maze model.

[0067] Male Long Evans rats (180-400 g) or male NIH Swiss mice (18-35 g)were obtained from Harlan Sprague-Dawley, Cumberland, Ind., USA andacclimated at least 3 days before testing. Animals were housed at 23±2°C. (relative humidity 30% to 70%) and given Purina Certified Rodent Chowand water ad libitum. The photoperiod was 12 hours of light and 12 hoursof dark, with dark onset at approximately 1800 hours.

[0068] Test compounds were dissolved in a vehicle of purified water andneutralized with 5 N NaOH to a pH of 7-8 when applicable. Diazepam(Sigma Chemical Company, St. Louis, Mo.) was suspended in purified waterby the dropwise addition of Tween 80. Control animals received therespective vehicle.

[0069] SL-LAB (San Diego Instruments, San Diego, Calif.) chambers wereused for conditioning sessions and for the production and recording ofstartle responses. A classical conditioning procedure was used toproduce potentiation of startle responses. Briefly, on the first 2 days,rats were placed into dark startle chambers in which shock grids wereinstalled. Following a 5-minute acclimation period, each rat received a1 mA electric shock (500 ms) preceded by a 5 second presentation oflight (15 watt) which remained on for the duration of the shock. Tenpresentations of the light and shock were given in each conditioningsession, rats were gavaged with a solution of test compound of water andstartle testing sessions were conducted. A block of 10 consecutivepresentations of acoustic startle stimuli (110 dB, non-light-paired)were presented at the beginning of the session in order to minimize theinfluences of the initial rapid phase of habituation to the stimulus.This was followed by 20 alternating trials of the noise alone or noisepreceded by the light. Excluding the initial trial block, startleresponse amplitudes for each trial type (noise-alone vs. light+noise)were averaged for each rat across the entire test session.

[0070] As shown in the first row of Table 2, below, when given orally tofed rats, compounds of the present invention were active in the ratfear-potentiated startle test at 300 times lower doses when compared toLY354740. If this in vivo animal model data directly predicts humananxiety responses, compounds of the present invention would produceanxiolytic effects in humans at 300 fold lower doses than the parentcompound. Furthermore, the ability to produce a longer duration at lowerdoses when compared to parent may allow for once-a day dosing, asopposed to twice a day dosing.

In Vivo Exposure as Measured by Rat Plasma Concentration

[0071] To study the in vivo exposure of LY354740 following oral dosingof compounds of the present invention in comparison to LY354740, studiesmeasuring the plasma concentrations of LY354740 in rats were performed.

[0072] Mature Fischer 344 male rats (190-270 gram) were obtained fromHarlan Sprague-Dawley, Cumberland, Ind., USA and acclimated in the studyhousing for 3 days. On day 4, test compounds were dissolved in bufferedwater (1 mg/ml=test compound/20 mM potassium dihydrogen phosphate, pH=2)and given orally as a single 5mg/kg dose. Blood samples were collectedthrough orbital sinus or cardiac puncture (last time point) at 0.5 and 1hour or, alternatively, 1 and 3 hours. Plasma samples were stored at−20° C. in the presence of phenylmethylsulfonyl fluoride, a proteaseinhibitor, prior to analysis. Plasma samples and internal standardcompounds were pretreated by solid phase extraction (SAX support,methanol/water/dilute acetic acid). As shown in the second row of Table2, below, the plasma concentrations (ng/ml) of LY354740 for each testcompound were determined by LC/MS/MS and are presented as a sum of theconcentrations at the 0.5 and 1 hour or, alternatively, 1 and 3 hoursample time points. TABLE 2 Comparison LY354740 and compounds of thepresent invention in the rat fear-potentiated startle assay ParameterCompound Measured LY354740 formula I MED (1 hour 3.0 mg/kg p.o. 0.01mg/kg p.o. pre- treatement) Rat Exposure 466 ng/ml 7114 ng/ml (ng/ml ofLY354740 following 5 mg/kg p.o.)

[0073] As shown above in Tables 1 and 2, in vitro studies show that thecompounds of the present invention had no appreciable affinity per sefor mGlu2/3 receptors. This indicates that the in vivo pharmacology ofthis compound in rats and humans would likely reflect the conversion ofthe prodrug to the parent molecule, LY354740, which then acts at mGlu2/3receptors to produce a therapeutic effect. Further, in fact, when givenorally to rats, the compounds of the current invention exhibit a 15 foldincrease in plasma concentration of LY354740. when compared to LY354740.This demonstrates compounds of the present invention are converted toLY354740 in vivo.

[0074] The compounds of the present invention are preferably formulatedprior to administration. Therefore, another aspect of the presentinvention is a pharmaceutical formulation comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier, diluent, or excipient. Thepharmaceutical formulations may be prepared by procedures well-known byone of ordinary skill in the art. In making the compositions of thepresent invention, the active ingredient will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier, and maybe in the form of a capsule, sachet, paper, or other container. When thecarrier serves as a diluent, it may be a solid, semi-solid, or liquidmaterial which acts as a vehicle, excipient, or medium for the activeingredient. The compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols, ointments containing, for example, up to10% by weight of active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

[0075] Some examples of suitable carriers, excipients, and diluentsinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum,acacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water syrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc,magnesium stearate, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents, or flavoring agents.Compositions of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart.

[0076] The compositions are preferably formulated in a unit dosage form,each dosage containing from about 5 mg to about 500 mg activeingredient, preferably about 25 mg to about 300 mg active ingredient. Asused herein the term “active ingredient” refers to a compound includedwithin the scope of formula I.

[0077] The term “unit dosage form” refers to a physically discrete unitsuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical carrier, diluent, or excipient.

[0078] The following Examples further illustrate the compounds of thepresent invention and the methods for their synthesis. The Examples arenot intended to be limiting to the scope of the invention in anyrespect, and should not be so construed. All experiments were run undera positive pressure of dry nitrogen or argon. All solvents and reagentswere purchased from commercial sources and used as received, unlessotherwise indicated. Dry tetrahydrofuran (THF) was obtained bydistillation from sodium or sodium benzophenone ketyl prior to use.Proton nuclear magnetic resonance (¹H NMR) spectra were obtained on aBruker Avance II bay-500 at 500 MHz, a Bruker Avance I bay-200 at 200MHzor a Varian Inova at 500 MHz. Electrospray mass spectroscopy (ESI) wasperformed on a Agilent MSD/B intrument using acetonitrile/aqueousammonium acetate as the mobile phase. Free atom bombardment massspectroscopy (FABMS) was performed on a VG ZAB-2SE instrument. Fielddesorption mass spectroscopy (FDMS) was performed using either a VG 70SEor a Varian MAT 731 instrument. Optical rotations were measured with aPerkin-Elmer 241 polarimeter. Chromatographic separation on a WatersPrep 500 LC was generally carried out using a linear gradient of thesolvents indicated in the text. The reactions were generally monitoredfor completion using thin layer chromatography (TLC). Thin layerchromatography was performed using E. Merck Kieselgel 60 F₂₅₄ plates, 5cm×10 cm, 0.25 mm thickness. Spots were detected using a combination ofUV and chemical detection (plates dipped in a ceric ammonium molybdatesolution [75 g of ammonium molybdate and 4 g of cerium (IV) sulfate in500 mL of 10% aqueous sulfuric acid] and then heated on a hot plate).Flash chromatography was performed as described by Still, et al. Still,Kahn, and Mitra, J. Org. Chem., 43, 2923 (1978). Elemental analyses forcarbon, hydrogen, and nitrogen were determined on a Control EquipmentCorporation 440 Elemental Analyzer, or were performed by the UniversidadComplutense Analytical Centre (Facultad de Farmacia, Madrid, Spain).Melting points were determined in open glass capillaries on a Gallenkamphot air bath melting point apparatus or a Büchi melting point apparatus,and are uncorrected.

[0079] The abbreviations, symbols and terms used in the examples havethe following meanings.

[0080] Ac=acetyl

[0081] Anal.=elemental analysis

[0082] Bn or Bzl=benzyl

[0083] Bu=butyl

[0084] BOC=butoxycarbonyl

[0085] calcd=calculated

[0086] D₂O=deuterium oxide

[0087] DCC=dicyclohexylcarbodiimide

[0088] DIBAL-H=diisobutyl aluminum hydride

[0089] DMAP=dimethylaminopyridine

[0090] DMF=dimethylformamide

[0091] DMSO=dimethylsulfoxide

[0092] EDC=N-ethyl-N′N′-dimethylaminopropyl carbodiimide

[0093] Et=ethyl

[0094] EtOH=ethanol

[0095] FAB=Fast Atom Bombardment (Mass Spectrascopy)

[0096] FDMS=field desorption mass spectrum

[0097] HOAt=1-hydroxy-7-azabenzotriazole

[0098] HOBt=1-hydroxybenzotriazole

[0099] HPLC=High Performance Liquid Chromatography

[0100] HRMS=high resolution mass spectrum

[0101] i-PrOH=isopropanol

[0102] IR=Infrared Spectrum

[0103] L=liter

[0104] Me=methyl

[0105] MeOH=methanol

[0106] MPLC=Medium Pressure Liquid Chromatography

[0107] Mp=melting point

[0108] MTBE=t-butyl methyl ether

[0109] NBS=N-bromosuccinimide

[0110] NMR=Nuclear Magnetic Resonance

[0111] Ph=phenyl

[0112] p.o.=oral administration

[0113] i-Pr=isopropyl

[0114] Rochelle's Salt=potassium sodium tartrate

[0115] SM=starting material

[0116] TBS=tert-butyldimethylsilyl

[0117] TEA=triethylamine

[0118] Temp.=temperature

[0119] TFA=trifluoroacetic acid

[0120] THF=tetrahydrofuran

[0121] TLC=thin layer chromatography

[0122] t-BOC=tert-butoxycarbonyl

Preparation 1 Synthesis of(1S,2S,5R,6S)-2-tert-Butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

[0123]

[0124] A 1 L flask was charged with(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (24.4 g, 0.12 mol, 1 equiv), dioxane (200 mL) anddi-tert-butyl dicarbonate (52.4 g, 0.24 mol, 2.0 equiv). The suspensionwas vigorously stirred while 1N sodium hydroxide (420 mL, 3.5 equiv) wasadded. The mixture was stirred for 2 days, then 2.0 more equiv ofdi-tert-butyl dicarbonate were added and the reaction stirred for 3additional days at room temperature. After 5 total days of reaction,water (400 mL) was added to dissolve the salts. The aqueous layer wasextracted with ethyl acetate (4×100 mL) and acidified to pH 2 with 6 Nhydrochloric acid. The acidic aqueous phase was extracted with ethylether (6×200 mL). The combined ether extracts were washed with water(250 mL) and brine (250 mL). After drying over sodium sulfate, solventswere evaporated under vacuum to afford a foamy white solid (26.4 g).

[0125] 77% Yield; mp 100-101° C. [α]_(D) ²⁵=41.1° (c=1.0, MeOH). ¹H NMR(Methanol-d₄) δ: 4.98 (brs, 1H), 2.44 (dd, 1H, J=6.2, 2.6 Hz), 2.19-1.92(m, 4H), 1.62 (t, 1H, J=2.8 Hz), 1.43 (s, 9H), 1.29 (m, 1H). ¹³C NMR(Methanol-d₄) δ: 175.6, 175.2, 158.2, 60.1, 34.6, 31.9, 28.4, 27.2,25.6, 20.6. MS (Electrospray): 285.12.

Preparation 2 Synthesis of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride

[0126]

[0127] (1S, 2S, 5R,6S)-2-tert-Butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (20 g, 0.07 mol, 1.0 equiv) was dissolved in 210 ml of drydimethylformamide and potassium carbonate (21.3 g, 0.154 mol, 2.2 equiv)was added at 0° C. under nitrogen. After 15 minutes, methyl iodide (17.6ml, 0.28 mol, 4.0 equiv) was added. The reaction mixture was warmed upslowly and stirred at room temperature for 3 h. Water (200 ml) was addedand the aqueous phase was extracted with ethyl ether (4×75 ml each). Thecombined organic phase was washed with cold water (4×50 ml), and theaqueous phase extracted again with ethyl ether (2×50 ml). After dryingthe organic phase over sodium sulfate and evaporating under vacuum, afoamy solid((1S,2S,5R,6S)-2-tert-butoxycarbonylamino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid-2,6-dimethyl ester) was obtained (19.2 g, 87% yield).

[0128] This compound was diluted with 150 ml of a saturated solution ofhydrogen chloride gas in ethyl acetate and the mixture vigorouslystirred for 1 hour (a white precipitate appeared within 15 minutes). Thesolid was filtered, rinsed with ethyl ether and thoroughly dried underhigh vacuum.

[0129] 73% Yield; mp 193-194° C. [α]_(D) ²⁵=+22.2° (c=1.0, MeOH). ¹H NMR(D₂O) δ: 3.86 (s, 3H), 3.67 (s, 3H), 2.31-2.04 (m, 6H), 1.57 (m, 1H).¹³C NMR (Methanol-d₄) δ: 171.9, 170.2, 65.6, 52.8, 51.2, 32.4, 29.9,28.5, 26.2, 20.7.

Alternative Synthesis of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride

[0130]

[0131] Thionyl chloride (807 mL, 11.1 mol) was added to methanol (9.5 L)over a period of 1 h while maintaining the temperature between 2-20° C.The solution was maintained for 30 min, then(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (1.61 kg, 7.92 mol) was added. The resulting solution washeated to 47° C. and maintained between 47-50 □C for 17 h. Approximately7.3 L of methanol was then removed by vacuum distillation (47-50° C.,240-275 mm Hg). The remaining methanol was removed by azeotropicdistillation with t-butyl methyl ether (MTBE) at atmospheric pressure[added MTBE (10 L), removed 8.5 L; added MTBE (10 L), removed 8.5 L;added MTBE (8 L), removed 5.1 L]. During the course of the distillationsa white solid began to precipitate from the solution. After completionof the distillations, MTBE (2 L) was added to the resulting slurry, andthe slurry was cooled to 22° C. The solid was filtered, rinsed with MTBE(2 L) and dried under vacuum to afford 1.94 kg (98%) of the titlecompound as a white solid.

[0132] Analysis Calculated for C₁₀H₁₆NO₄Cl: C, 48.10; H, 6.46; N, 5.61;Cl, 14.20. Found: C, 47.88; H, 6.25; N, 5.57; Cl, 14.52.

General Procedure for the Coupling Reaction of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride with N-BOC-(L)-aminoacids

[0133] The starting dimethyl ester hydrochloride salt (1.0 equiv), theproduct of Example Preparation 2, was suspended in dry dichloromethane(0.1 M solution) under nitrogen. The corresponding N-BOC-aminoacid (1.5equiv), N-ethyl-N′,N′-dimethylaminopropylcarbodiimide (EDC, 1.5 equiv)and 1-hydroxybenzotriazole (HOBt, 1.5 equiv) were added in one portion,followed by triethyl amine(1.0 equiv) via syringe and, finally,dimethylaminopyridine (DMAP, 0.1 equiv). The reaction mixture wasstirred overnight at room temperature, then hydrolyzed by addition of INhydrochloric acid (20 ml/mmol) and diluted with methylene chloride (10ml/mmol). The aqueous layer was extracted with methylene chloride (5ml/mmol) and the combined organic layers washed twice with 1 Nhydrochloric acid (10 ml/mmol), and finally with water and brine (10ml/mmol each). After drying over sodium sulfate and evaporation undervacuum the crude residue was purified by silica gel chromatography usingthe appropriate eluent (typically mixtures hexanes/ethyl acetate).

Alternative Procedure for the Coupling Reaction of(1S,2S,5R,6S)-2-Amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddimethyl ester hydrochloride with N-BOC-aminoacids

[0134] A solution of dicyclohexylcarbodiimide (DCC) (1.1 equiv) inmethylene chloride (4.0 M solution) was added to a mixture ofPreparation 2 (1.0 equiv), triethylamine (1.0 equiv) andN-t-butoxycarbonyl-L-alanine (1.1 equiv) in methylene chloride (1.0 Msolution) over a period of approximately 1.5 h while stirring. Theresulting mixture was stirred for 1-12 h then filtered. The filter cake(dicyclohexylurea) was rinsed with methylene chloride, and the filtratewas washed with 0.1 M NaHCO₃ followed by 1.0 N hydrochloric acid. Theorganic phase was dried (Na₂SO₄), filtered and concentrated to affordthe title compound as an oil.

EXAMPLE 1 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester

[0135]

[0136] The starting dimethyl ester hydrochloride salt (1.0 equiv), theproduct of Preparation 2, was suspended in dry dichloromethane (0.1 Msolution) under nitrogen. N-BOC-(L)-alanine (1.5 equiv),N-ethyl-N′,N′-dimethylaminopropylcarbodiimide (EDC, 1.5 equiv) and1-hydroxybenzotriazole (HOBt, 1.5 equiv) were added in one portion,followed by triethyl amine(1.0 equiv) via syringe and, finally,dimethylaminopyridine (DMAP, 0.1 equiv). The reaction mixture wasstirred overnight at room temperature, then hydrolyzed by addition of 1Nhydrochloric acid (20 ml/mmol) and diluted with methylene chloride (10ml/mmol). The aqueous layer was extracted with methylene chloride (5ml/mmol) and the combined organic layers washed twice with 1 Nhydrochloric acid (10 ml/mmol), and finally with water and brine (10ml/mmol each). After drying over sodium sulfate and evaporation undervacuum the crude residue was purified by silica gel chromatography usingmixtures of hexanes/ethyl acetate.

[0137] 50% Yield. Foamy white solid. mp 51-52° C. [α]_(D) ²⁵=27.7(c=0.52, CHCl₃) ¹H NMR (CDCl₃) δ: 7.28 (brs, 1H), 5.04 (brd, 1H, J=7.6Hz), 4.16 (m, 1H), 3.74 (s, 3H), 3.66 (s, 3H), 2.49 (dd, 1H, J=13.9, 8.3Hz), 2.42 (dd, 1H, J=6.3, 2.8 Hz), 2.18-1.89 (m, 3H), 1.70 (t, 1H, J=2.9Hz), 1.45 (s, 9H), 1.33 (d, 3H, J=7.0 Hz), 1.19 (m, 1H). ¹³C NMR (CDCl₃)δ: 172.8, 172.6, 172.6, 155.7, 80.2, 66.3, 52.6, 51.8, 49.5,.34.4, 32.0,28.2, 28.1, 26.6, 21.1, 17.6.

Alternative Synthesis of(1S,2S,5R,6S)-2-1(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester

[0138] A solution of dicyclohexylcarbodiimide (DCC) (1.1 equiv) inmethlyene chloride (4.0 M solution) was added to a mixture of ExamplePreparation 2 (1.0 equiv), triethylamine (1.0 equiv) andN-t-butoxycarbonyl-L-alanine (1.1 equiv) in methlyene chloride (1.0 Msolution) over a period of approximately 1.5 h while stirring. Theresulting mixture was stirred for 1-12 h then filtered. The filter cake(dicyclohexylurea) was rinsed with methlyene chloride, and the filtratewas washed with 0.1 M NaHCO₃ followed by 1.0 N hydrochloric acid. Theorganic phase was dried (Na₂SO₄), filtered and concentrated to affordthe title compound as an oil.

EXAMPLE 2 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

[0139]

[0140] A solution of 2 M NaOH (5.45 L, 10.9 mol) was added to a solutionof(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid dimethyl ester (4.52 mol, crude) in THF (2.8 L). The resultingmixture was stirred at ambient temperature for 3 h then extracted withCH₂Cl₂ (2×3 L). Ethyl acetate (5 L) and tetrahydrofuran (3 L) were thenadded to the aqueous phase. While stirring, concentrated HCl (970 mL)was added to the mixture until the pH=2. The organic phase was dried(MgSO₄) and filtered. The aqueous phase was then extracted with ethylacetate (5 L). The organic phase was dried (MgSO₄), filtered andcombined with the previous organic phase. The combined organics wereconcentrated to a soft solid. Ethyl acetate was then added, and themixture was concentrated to a soft solid. Ethyl acetate (3.5 L) wasagain added. The mixture was concentrated until a freely flowingsuspension was present. Heptane ( 1.8 L) was then added, and the slurrywas stirred at ambient temperature for 15 h. The solid was filtered,washed with heptane (3 L) then dried under vacuum to afford the titlecompound.

[0141] Yield 1.36 kg (84%) as an approximate 85:15 mixture of rotamersas a white solid. [α]_(D)25-24.8(C1.0, MeOH) ¹H NMR (DMSO-d₆) δ 12.20(s, 2 H), 8.40 (s, 0.85 H), 8.36 (s, 0.15 H), 6.69 (d, J=8.2 Hz, 0.85H), 6.33 (br d, 0.15 H), 3.99 (quintet, J=7.2 Hz, 0.85 H), 3.84 (br m,0.15 H), 2.18-2.13 (m, 2 H), 1.91-1.84 (m, 1 H), 1.82-1.75 (m, 2 H),1.46 (br s, 0.85 H), 1.43 (br s, 0.15 H), 1.35 (s, 9 H), 1.23-1.15 (m, 1H), 1.13 (d, J=6.9 Hz, 3 H). ¹³C NMR (CD₃OD) δ 176.4, 176.0 (2 C),157.5, 80.5, 67.3 (minor rotamer), 67.2 (major rotamer), 50.9, 35.6,32.8, 29.3, 28.7, 27.4, 22.1, 18.5. MS (EI) calcd for C₁₆H₂₈N₃O₇ (M+NH₄⁺) 374.20, found 374.24 m/z.

Alternative Synthesis of (1S,2S,5R,6S) -2-[(2′S)-(2′-tert-Butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

[0142]

[0143] A solution of(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acidmonohydrate (85 g, 418 mmol) and MeOH (850 mL) was cooled to 10° C.Thionyl chloride (199 g, 1.67 mol) was added at a rate such that thetemperature did not exceed 20° C. The solution was then heated to 50° C.and stirred for 6 h. Upon completion of the reaction, the solution wascooled to room temperature and concentrated to approximately 170 mLtotal volume under reduced pressure at 20-30° C. Water (850 mL) wasadded, and the pH of the solution was adjusted to approximately pH 2.0with 1.0 N NaOH (300 mL). The solution was concentrated under reducedpressure until the temperature reached approximately 40° C. Methylenechloride (850 mL) was then added, and the pH of the solution wasadjusted to pH 8 with 1.0 N NaOH (180 mL). The phases were separated,and the aqueous phase was extracted with CH₂Cl₂ (425 mL). The combinedorganic phases containing the corresponding dimethyl ester wereconcentrated to approximately 425 mL total volume and held for furtherprocessing.

[0144] In a separate reaction vessel a solution ofN-t-butoxycarbonyl-L-alanine (83.2 g, 439 mmol) and 4-methylmorpholine(44.4 g, 439 mmol) in CH₂Cl₂ (712 mL) was cooled to −5-−10° C. Isobutylchloroformate (59.9 g, 439 mmol) was then added at rate such that thetemperature did not exceed −5° C. Upon completion of the addition, thesolution was stirred for 15 min. Simultaneously, CH₂Cl₂ (20 mL) wasadded to the dimethyl ester solution previously prepared, and thissolution was cooled to −5° C. The dimethyl ester solution (445 mL) wasthen added to the isobutyl mixed anhydride mixture. The cooling bath wasremoved, and the corresponding mixture-was stirred for 30 min. Asolution of 1.0 N HCl (445 mL) was then added. The phases wereseparated, and the organic phase was washed with 1.0 N HCl (445 mL). Theorganic phase was concentrated to approximately 180 mL total volume. THF(450 mL) was then added, and the resulting solution was concentrated toapproximately 180 mL total volume. To this solution was added 1.0 N NaOH(1.67 L, 1.67 mol). The resulting mixture was heated to 40° C., stirredfor 1.5 h then cooled to room temperature. Ethyl acetate (2.4 L) wasadded, and the pH of the aqueous phase was adjusted to pH 2.1 withconcentrated HCl (150 mL). The phases were separated, and the aqueousphase was extracted with ethyl acetate (800 mL). The combined organicphases were dried with MgSO₄, filtered and washed with EtOAc (2×320 mL).The resulting solution was then concentrated to approximately 400 mLtotal volume. Ethyl acetate (800 mL) was added, and the solution wasconcentrated to 400 mL). This ethyl acetate addition/concentration wasrepeated again, then heptane (640 mL) was added. The resulting mixturewas stirred for 2 h, filtered and washed with a 2:1 mixture ofheptane-ethyl acetate (2×320 mL) to afford 115.5 g (78% yield) of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid as a white solid.

EXAMPLE 3 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

[0145]

[0146] To a solution of ethyl acetate (500 mL) was added HCl (79.0 g,2.16 mol). The resulting HCl solution was then added to a slurry of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo(3.1.0]hexane-2,6-dicarboxylicacid (100 g, 281 mmol) in ethyl acetate (500 mL) at a rate such that thetemperature did not exceed 25° C. The resulting mixture was stirred for3.5 hours then filtered affording 82.6 g of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride as an amorphous, white solid. This white solid wasthen added to acetone (290 mL) and water (57 mL). The resulting mixturewas heated to 48-52° C., and water (6.4 mL) was added until all of thesolid dissolved. Acetone (2.2 L) was added to the resulting solutionover a period of approximately 1 h. When the addition of acetone began,the heating mantle was removed. After the addition was complete, themixture was cooled to 0-−10° C. and stirred for 4 h. The mixture wasthen filtered and washed with cold acetone (75 mL) affording(1S,2S,5R,6S)-2-[(2′S)-(2′-amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride that was dried under vacuum at 40° C. to provide76.6. g (93% yield) of the title compound as a white, crystalline solid.

[0147] 72% Yield. White crystalline solid. mp>250° C., dec. [α]_(D)²⁵=−7.80 (c=1.0, MeOH). ¹H NMR (Methanol-d₄) δ: 3.96 (q, 1H, J=7.0 Hz),2.47 (dd, 1H, J=6.3, 2.7 Hz), 2.37 (dd, 1H, J=13.6, 8.2 Hz), 2.18-1.92(m, 3H), 1.66 (t, 1H, J=3.0 Hz), 1.53 (d, 3H, J.=7.0 Hz), 1.46-1.34 (m,1H). ¹³C NMR (Methanol-d₄) δ: 175.2, 174.7, 170.2, 66.4, 49.0, 36.6,32.0, 28.5, 26.3, 21.2, 16.6. 80% Yield. White solid.

EXAMPLE 4 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methanesulfonate

[0148]

[0149] A solution of(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-butoxycarbonylamino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (1.07 g, 3.00 mmol), methanesulfonic acid (584 μL, 9.00 mmol) anddioxane (10 mL) was stirred for 48 h. The mixture was filtered and driedto afford (1S, 2S, 5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methane sulfonate as a crude, white, amorphous solid (1.05 g). Asample of this solid (1.0 g) was dissolved in MeOH (10 mL). The solutionwas concentrated to 3.3 g total weight and seed crystals were added.Ethyl acetate (10 mL) was then added to the mixture over a period of 15min. The mixture was stirred for 30 min, filtered and dried under vacuumto afford 830 mg of the title compound as a white, crystalline solid.

[0150] Yield 78% ¹H NMR (CD₃OD) δ 3.96 (q, J=7.1 Hz, 1 H) , 2.71 (s, 3H), 2.45 (dd, J=6.4, 2.7 Hz, 1 H), 2.38 (dd, J=13.9, 8.4 Hz, 1 H),2.20-2.08 (m, 1 H), 2.01-1.93 (m, 2 H), 1.67 (t, J=2.9 Hz, 1 H), 1.52(d, J=7.0 Hz, 3 H), 1.46-1.35 (m, 1 H) ¹³C NMR (CD₃OD) δ 176.3, 175.7,171.2, 67.4, 50.0, 39.5, 35.7, 33.1, 29.5, 27.4, 22.2, 17.6. Anal. Calcdfor C₁₂H₂₀N₂O₈S: C, 40.90; H, 5.72; N, 7.95. Found: C, 40.81; H,. 5.69;N, 7.83.

EXAMPLE 5 Synthesis of(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

[0151]

[0152] (1S, 2S, 5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride (1.0 g, 3.42 mmol) was dissolved in water (1 mL), and1.0 N NaOH (3.42 mL, 3.42 mmol) was added. The solution was maintainedin the refrigerator for 24 h. The solution remained clear. Acetone (2mL) was added, and the solution was stored in the refrigerator for 16 h.A white solid precipitated out of solution, and mixture could not bestirred. Acetone (4 mL) was added, and the mixture was stirred at rt,then filtered and dried to afford 630 mg of the title compound as awhite crystalline solid which contained 2-4% NaCl.

[0153] Yield 72% ¹H NMR (CD₃OD) δ 3.93 (q, J=7.1 Hz, 1 H) , 2.48 (dd, ,J=6.6, 2.9 Hz, 1 H), 2.32 (dd, , J=13.5, 8.4 Hz, 1 H), 2.20-2.08 (m, 1H), 2.01-1.90 (m, 2 H), 1.61 (t, , J=2.9 Hz, 1 H), 1.51 (d, , J=7.0 Hz,3 H), 1.48-1.33 (m, 1 H) ¹³C NMR (CD₃OD) δ 176.9 (2 C), 171.1, 68.0,50.1, 35.9, 33.2, 29.7, 27.3, 22.5, 17.6.

1. A compound of formula I

wherein R¹³, R¹⁴ and R¹⁷ are hydrogen; or a pharmaceutically acceptablesalt thereof.
 2. A pharmaceutically acceptable salt of a compound offormula I as claimed claim 1 which is an acid-addition salt made with anacid which provides a pharmaceutically acceptable anion or, for acompound which contains an acidic moiety, which is a salt made with abase which provides a pharmaceutically acceptable anion.
 3. Thepharmaceutically acceptable salt of a compound of formula I as claimedin claim 2 which is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride salt.
 4. The pharmaceutically acceptable salt of acompound of formula I as claimed in claim 2 which is(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-propionyl]amino-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid methane sulfonate salt.
 5. A pharmaceutical formulation comprisingin association with a pharmaceutically acceptable carrier, diluent orexcipient, a compound of formula I (or a pharmaceutically acceptablesalt thereof) as provided in any one of claims 1 to
 4. 6. A process forpreparing the compound of formula I, or a pharmaceutically acceptablesalt thereof, as claimed in any one of claims 1 to 4 comprising: for acompound of formula I, deprotecting a compound of formula IV

where R^(m) is a nitrogen protecting group; and R¹³ and R¹⁴ are carboxyprotecting groups; whereafter, for any of the above procedures, when afunctional group is protected using a protecting group, removing theprotecting group; whereafter, for any of the above procedures, when apharmaceutically acceptable salt of a compound of formula I is required,it is obtained by reacting the basic form of such a compound of formulaI with an acid affording a physiologically acceptable counterion, or,for a compound of formula I which bears an acidic moiety, reacting theacidic form of such a compound of formula I with a base which affords apharmaceutically acceptable cation, or by any other conventionalprocedure.
 7. A method for affecting the cAMP-linked metabotropicglutamate receptors in a patient, which comprises administering to apatient requiring modulated excitatory amino acid neurotransmission apharmaceutically-effective amount of a compound of any one of claims 1to
 4. 8. A method of administering an effective amount of a compound offormula II, where R¹³ and R¹⁴ are both hydrogen (a di-acid), whichcomprises administering to a patient requiring modulated excitatoryamino acid neurotransmission a pharmaceutically effective amount of acompound of any one of claims 1 to
 4. 9. A method for treating aneurological disorder in a patient which comprises administering to thepatient in need of treatment thereof a pharmaceutically-effective amountof a compound of any one of claims 1 to
 4. 10. The method of claim 9wherein said neurological disorder is cerebral deficits subsequent tocardiac bypass and grafting; cerebral ischemia; spinal cord trauma; headtrauma; Alzheimer's Disease; Huntington's Chorea; amyotrophic lateralsclerosis; AIDS-induced dementia; perinatal hypoxia; hypoglycemicneuronal damage; ocular damage and retinopathy; cognitive disorders;idiopathic and drug-induced Parkinson's Disease; muscular spasms;migraine headaches; urinary incontinence; drug tolerance, withdrawal,and cessation; smoking cessation; emesis; brain edema; chronic pain;sleep disorders; convulsions; Tourette's syndrome; attention deficitdisorder or tardive dyskinesia.
 11. The method of claim 10 wherein saidneurological disorder is drug tolerance, withdrawal, and cessation orsmoking cessation.
 12. A method for treating a psychiatric disorder in apatient which comprises administering to the patient in need oftreatment thereof a pharmaceutically-effective amount of a compound ofany one of claims 1 to
 4. 13. The method of claim 12 wherein saidpsychiatric disorder is schizophrenia, anxiety and related disorders,depression, bipolar disorders, psychosis or obsessive compulsivedisorders.
 14. The method of claim 13 wherein said psychiatric disorderis anxiety and related disorders.
 15. A compound of formula IV

wherein R^(m) is a nitrogen protecting group; R¹¹ is CO₂R¹⁴ and R¹² ishydrogen or fluoro; or R¹¹ is hydrogen or fluoro and R¹² is CO₂R¹⁴; andR¹³ and R¹⁴ are carboxy protecting groups; provided that when R¹² ishydrogen and R^(m) is C(O)CH₃, R¹³ and R¹⁴ are not both methyl.
 16. Acompound of formula IV as claimed in claim 21 wherein R^(m) istert-butoxycarbonyl, R¹¹ is CO₂R¹⁴; R¹³ and R¹⁴ are both methyl; and R¹²is hydrogen.
 17. A novel compound of formula I, or a pharmaceuticallyacceptable salt thereof, as claimed in any one of claims 1 to 4 for useas a pharmaceutical.
 18. Use of a compound of formula I, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for treating neurological or psychiatric disorders.
 19. Anovel compound of formula I substantially as hereinbefore described withreference to any of the Examples.
 20. A method for affecting theCAMP-linked metabotropic glutamate receptors in a mammal, whichcomprises administering to a mammal requiring modulated excitatory aminoacid neurotransmission a pharmaceutically effective amount of a compoundof formula I substantially as hereinbefore described with reference toany of the Examples.
 21. A process for preparing a novel compound offormula I substantially as hereinbefore described with reference to anyof the Examples.